Journal: PLoS Computational Biology
Article Title: Microarray Analysis of LTR Retrotransposon Silencing Identifies Hdac1 as a Regulator of Retrotransposon Expression in Mouse Embryonic Stem Cells
doi: 10.1371/journal.pcbi.1002486
Figure Lengend Snippet: (A) Plot showing the differential behaviour of different RLTR4 retrotransposon probe populations in Ring1B −/− single knockou t ES cells. Different RLTR4 probe populations are colour-coded as shown in the legend, and vertical lines indicate a 4 fold change. (B) qRT-PCR verification of repetitive element expression in Ring1B −/− ES cells. Expression levels (mean ± standard error) were normalized to β-Actin and expressed relative to wild-type control ES cells. MMERVK10C env.c and LINE1 5′UTR primer sets were used to assess MMERVK10C and LINE-1 expression. The asterisk indicates a statistically significant difference (p<0.05). Note that different primers for RLTR4 elements behave differently in the qRT-PCR assay. (C) qRT-PCR for different MMERVK10C primer sets in Tex19.1 −/− knockout and littermate control testes at 16 dpp. Expression levels (mean ± standard error for three animals) were normalized to β-Actin and expressed relative to littermate controls. Asterisks indicate statistically significant differences (p<0.05) (D) Plot showing the MMERVK10C genomic contigs flanked by RLTR10C LTRs that match only upregulated probes (blue), only unaffected probes (brown), neither class of probes (grey), or both classes of probe (green) in Tex19.1 −/− testes. Each contig is represented by a horizontal line that indicates the regions of the MMERVK10C sequence within it. The upregulated MMERVK10C contigs appear to contain recurrent deletions and may be non-autonomous. The positions of the qRT-PCR primers used in (C) are shaded orange. (E) Plot showing the bimodal behaviour of IAP-int retrotransposon probe populations in Dnmt TKO ES cells. Vertical lines indicate a 4 fold change. (F) qRT-PCR for of repetitive elements in Dnmt TKO ES cells. Expression levels (mean ± standard error) were normalized to Gapdh and expressed relative to wild-type control ES cells. The asterisk indicates a statistically significant difference (p<0.05). The LINE1 5′UTR.b primer set was used to assess LINE-1 expression. Note the difference in behaviour between the two IAP-int primer sets. The IAP contig carrying deletions in the AP-1 binding site shown in panel G (IAP_chr10 primers) is expressed but not upregulated in Dnmt TKO ES cells. (G) Sequence alignment between an LTR of a full-length IAP element that does not change expression in Dnmt TKO ES cells (IAP_chr10), and the consensus sequence for the LTR (IAPLTR1a_Mm). The 10 bp deletion removes the AP-1 transcription factor binding site in the LTR.
Article Snippet: Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data.
Techniques: Quantitative RT-PCR, Expressing, Control, Knock-Out, Sequencing, Binding Assay
